Identification of mast cell specific receptor
crucial for pseudoallergic drug reactions
Xinzhong Dong
Dept. of Neuroscience
Johns Hopkins University
HHMI
Disclosure
In relation to this presentation, I declare that there are no
conflicts of interest.
A conflict of interest is any situation in which a speaker or immediate family members have interests, and those may cause a conflict
with the current presentation. Conflicts of interest do not preclude the delivery of the talk, but should be explicitly declared. These
may include financial interests (eg. owning stocks of a related company, having received honoraria, consultancy fees), research
interests (research support by grants or otherwise), organisational interests and gifts.
Distribution of mast cells
•
Found in nearly all tissues but enriched in regions close to the interface
between the body and the environment (skin, GI, lung)
Nature Reviews Immunology 4 (2004), 787-799.
Mast cells secret many substances
– Secrete many pro-inflammatory mediators
– IgE-dependent and independent activation pathways
IgE
inflammation
Nature 420 (2002), 875-878.
IgE-independent mast cell activation
• IgE-independence
– Can’t detect IgE antibody titer to the substance
– Doesn’t require prior exposure to the substance
• Evokes similar allergic symptoms
− Mild reactions: flushing, pruritus, rash
− Severe reactions: hives, bronchospasm, hypotension,
body temperature drop
• “Pseudoallergic” reactions
• Critical for human health but poorly understood
Inducers of IgE-independent mast cell activation
• Numerous endogenous and exogenous peptides/substances
• A common chemical structure: a combination of bulky hydrophobic and
cationic moieties in close proximity
• Collectively called basic secretagogues
Natural peptides that activate mast cells
•
•
•
•
•
•
•
•
•
•
•
•
•
Substance P
Bradykinin
Cortistatin
Defensins
Cathelicidin, LL-37
PAMP
PACAP
ACTH fragments
Brain Natriuretic Peptide
VIP
NPY
Mastoparan (wasp venom)
etc…
Compound 48/80 induces IgE-independent mast
cell degranulation
• It is the most frequently used mast cell degranulator
(~2400 citations)
• It degranulates mast cells in a IgE-independent manner.
• It causes mast cells to release histamine, serotonin,
proteases, TNFα, interleukins, etc.
(Kulka et al, 2008 Immunology; Gibbs et al, 2001 Exp Dematol; Krop et al 2010 Eur J
Pharmacol; Lai et al , Wolber et al, 2004 Eur J Pharmacol)
What is the molecular target?
Mas- related Genes (Mrg) encode a
family of G Protein-Coupled Receptors
Dong et al. Cell 2001
Some Mrgs are itch receptors
β-ala
proteases
MrgprD
?
Is Mrg expressed in mast cells?
Liu et al., Cell 2009
Liu et al, Science Sig. 2011
Liu et al. J. Neurosci. 2012
Han et al, Nat. Neuro. 2013
MrgB2 is exclusively expressed in
mouse mast cells
RT-PCRs to detect mRNAs of
different Mrgs in mast cells
MrgA1 2
MrgB1 2
3
3
4
4
6
5
Staining of mast cells in different tissues
Glabrous skin
Hairy skin
9 10 12 14 16 18 19
6
8 10 11 C11
Neg.
avidin
MrgB2
Trachea
Heart
Avidin: a mast cell marker
McNeil et al. Nature 2015
MrgX2/B2 is exclusively expressed in mast cells
• MrgX2 was exclusively active in human MCs ex vivo and exhibited the
highest fold change among candidates. (Motakis, et al Blood 2014)
MrgX2/B2 is also one of the highest expressing genes in mast cells.
MrgB2
Mouse
Genechip data (GeneAtlas GNF)
Mast cell degranulator compound 48/80 activates
MrgX2/B2 in HEK cells
Human X2
Time (sec)
Mouse B2
Time (sec)
Ca2+ imaging (each trace represents Ca2+ level in individual cells)
Mast cell degranulators specifically activate human
MrgX2 and mouse MrgB2
Human
MrgX2
Human
MrgX1
Mouse
MrgB2
Mouse Mouse
MrgB1 MrgB10
Mouse
MrgB11
PAMP

/




Compound 48/80






Cortistatin






Substance P






Chloroquine






EC50s of MrgX2/B2 activating substances
Substance
MrgB2 EC50
MrgX2 EC50
Compound 48/80
Substance P
Cortistatin-14
PAMP (9-20)
Mastoparan
Icatibant
Cetrorelix
Sermorelin
Octreotide
Leuprolide
Atracurium
Rocuronium
Ciprofloxacin
Moxifloxacin
Levofloxacin
Ofloxacin
3.7 ± 0.5 µg/ml
54.3 ± 4.9 µM
21.3 ± 0.9 µM
12.4 ± 1.6 µM
24.0 ± 3.6 µM
32.5 ± 2.0 µg/ml
23.4 ± 1.4 µg/ml
29.1 ± 1.2 µg/ml
10.0 ± 1.1 µg/ml
152.0 ± 7.1 µg/ml
44.8 ± 1.4 µg/ml
22.2 ± 3.3 µg/ml
126.5 ± 5.1 µg/ml
14.1 ± 2.1 µg/ml
807.6 ± 47.1 µg/ml
225.0 ± 25.4 µg/ml
470.1 ± 139.6 ng/ml
152.3 ± 48.0 nM
106.7 ± 39.3 nM
166.0 ± 35.7 nM
3.9 ± 0.7 µM
15.8 ± 2.7 µg/ml
221.7 ± 63.1 ng/ml
4.5 ± 0.9 µg/ml
6.6 ± 0.7 µg/ml
9.1 ± 0.7 µg/ml
28.6 ± 2.4 µg/ml
261.3 ± 14.4 µg/ml
6.8 ± 0.5 µg/ml
9.9 ± 0.6 µg/ml
22.7 ± 0.4 µg/ml
30.1 ± 1.5 µg/ml
MrgX2 or B2-HEK293 cells; Ca2+ imaging
Generated MrgB2 knockout mice by zinc finger
nuclease (ZFN)
Mouse #1: 4-bp deletion leads to a premature
translation stop in the 1st transmembrane domain.
MrgB2 is not required for mast cell survival and migration
avidin
1.2
Hairy skin
Mast cell/µm 2
Mast cell/µm 2
0.4
0.3
0.2
0.1
MUT
Glabrous skin
0.8
0.6
0.4
0.2
0
0
WT
MUT
4
1
% of total cells
WT
WT
MUT
Peritoneal
3
2
1
0
WT
MUT
Peritoneal mast cell degranulation
Response to Compound 48/80 (10 µg/ml)
MrgB2 KO mast cells exhibit normal IgEmediated activation
WT
Response to anti-IgE Ab
KO
48/80 response requires MrgB2
WT
Ca2+ imaging (Fluo-4);
Response to Compound 48/80 (10 µg/ml)
KO
48/80 response requires MrgB2
McNeil et al. Nature 2015
48/80-induced histamine release requires MrgB2
48/80-induced airway contraction
requires MrgB2
48/80-induced edema requires MrgB2
Tail vein injection of Evans Blue to detect
extravasation of blood vessels
IgE-dependent edema does not require MrgB2
MrgB2 mediates endogenous peptide induced
mast cell activation
Histamine release (pg/cell)
6
Histamine release (pg/cell)
MrgB2 is essential for IgE-independent
mast cell activation
8
6
WT
MUT
5
5
4
4
3
3
p<0.005
2
1
p<0.05
2
1
0
0
0
5
10
Compound 48/80 (µg/ml)
15
6
0
20
40
60
80
Mastoparan (µM)
12
2.5
10
2
2
p<0.05
4
50
100 150 200
Atracurium (µg/ml)
250
1
0
0
0
100 150 200 250
Icatibant (µg)
0.5
2
0
50
N.S.
1.5
6
p<0.0001
p<0.005
0
100
8
4
7
6
5
4
3
2
1
0
0
100 200 300 400
Ciprofloxcin (µg/ml)
500
0
10
20
30
40
50
Anti-IgE antibody (µg/ml)
MrgB2 does not disturb other GPCR signaling
FDA-approved peptidergic drug and
pseudoallergic reactions
• Many available, in use to treat a variety of disorders
• The majority are administered subcutaneous
• A number of them are linked to significant adverse
reactions at the injection site and (rare) reports of
anaphylaxis
FDA approved peptides administered SC or IM
Associated with injection
site reactions
• Icatibant (Bradykinin 2
receptor antagonists)
– angioedema
• Cetrorelix (GnRH agonists
and antagonists)
– Fertility treatments to
cancer
• GHRH and its variants
– Metabolic disorders and
HIV associated
lipodystrophy
• Octreotide (Somatostatin
analogs)
– Acromegaly, severe
diarrhea, cancer
Not associated with
injection site reactions
•
•
•
•
•
Vasopressin
Glucagon
Oxytocin
Secretin
Insulin
Injection site reactions to icatibant
A bradykinin receptor 2 antagonist
to treat hereditary angioedema
MrgB2 mediates peptide drug induced
mast cell activation
McNeil et al. Nature 2015
Icatibant in vivo response is almost entirely
absent in MrgB2 KO mice
Common chemical motif in 48/80 and small
molecule drugs
Neuromuscular blocking drugs (NMBDs)
THIQ
NMBDS are responsible for ~60% of
allergic reactions in a surgical setting
Fluoroquinolone
antibiotics
MrgB2 mediates small molecule drug induced
mast cell activation
Ciprofloxacin induced anaphylactic shock is
absent in MrgB2 KO mice
Human mast cell degranulation
Human MrgX2 is essential for IgE-independent
mast cell degranulation
Summary
• MrgB2/X2 is exclusively expressed in mast cells.
• MrgB2/X2 is the sole receptor for many basic secretagogues.
• MrgB2/X2 is not involved in IgE-dependent mast cell activation.
• MrgB2 mediates drug-induced anaphylactoid side effects.
Acknowledgements
Ben McNeil
Priyanka Pundir
Dustin Green
Jimmy Meixiong
Liang Han
Qin Liu
Andrew Kim
Zongxiang Tang
Yixun Geng
Kush Patel
Hao-Jui Weng
Yu-shin Kim
Zhe Li
Shuohao Sun
Colleen LaVinka
Kyoungsook Park
Yan Wang
Collaborators
JHU
Bradley Undem
National Institute for
Nanotechnology, Canada
Marianna Kulka
Priyanka Pundir
Funding: NIH; HHMI